EP3750508B1 - Method and device for the in-vitro investigation of the interaction between blood and a test object - Google Patents

Description

The invention relates to a method for in-vitro examination of the interaction of blood with a test object, in particular a cardiovascular implant or a medical device. In addition, a device for in-vitro examination of the interaction of blood with a test object is provided.

1. [1] W. van Oeveren, I.F.J. Tielliu, J. de Hart in "International Journal of Biomaterials", Bd. 2012, Article ID 673163 (Mai 2012 );
2. [2] W. van Oeveren in "Scientifica", Bd. 2013, Article ID 392584, S. 1-14 (2013 );
3. [3] U. Streller, C. Sperling, H. Hübner, R. Hanke, C. Werner in "Journal of Biomedical Materials Research Part B Applied Biomaterials", Bd. 66B, S. 379-390 (2003 );
4. [4] J. W. Mulholland, J. C. Shelton, X. Y. Luo in "Journal of Fluids and Structures", Bd. 20, Nr. 1, S. 129-140 (January 2005 );
5. [5] G. E. Engels, S. L. J. Blok, W. van Oeveren in "Biointerphases", Bd. 11, Nr. 3, 031004 (September 2016 );
6. [6] P. Borgdorff, R. H. van den Berg, M. A. Vis, G. C. van den Bos, G. J. Tangelder in "The Journal of Thorac Cardiovascular Surgery", Bd. 118, Nr. 5, S. 946-52 (1999 );
7. [7] A. B. Chandler in "Laboratory Investigation J Tech Methods Pathol." Bd. 7, Nr. 2, S. 110-114 (1958 );
8. [8] S. Braune, M. Grunze, A. Straub, F. Jung in "Biointerphases", 8:33, (Dezember 2013 );
9. [9] S. Braune, A. Lendlein, F. Jung in "Hemocompatibility of Biomaterials for Clinical Applications", 1. Auflage, Kapitel: Teile 1-3, S. 51-76 (November 2017 );
10. [10] S. Sinn, T. Scheuermann, S. Deichelbohrer, G. Ziemer, H. P. Wendel in "Journal of Materials Science: Materials in Medicine", Bd. 22, Nr. 6, S. 1521-1528 (Juni 2011 );
11. [11] WO 2005/033671 A1 ;
12. [12] JP 2007/089974 A ;
13. [13] US 4,627,419 A ;
14. [14] US 6,066,085 A ;
15. [15] US 2010/0204539 A1 ;
16. [16] US 6,579,223 B2 ; und
17. [17] M. Kilani et al. in "Sensors and Actuators" A 247 ( 2016 ) 440-447 .

The following prior art documents are cited in this description of the invention:

1. [1] W. van Oeveren, IFJ Tielliu, J. de Hart in "International Journal of Biomaterials", Vol. 2012, Article ID 673163 (May 2012 );
2. [2] W. van Oeveren in "Scientifica", Vol. 2013, Article ID 392584, pp. 1-14 (2013 );
3. [3] U. Streller, C. Sperling, H. Hübner, R. Hanke, C. Werner in "Journal of Biomedical Materials Research Part B Applied Biomaterials", Vol. 66B, pp. 379-390 (2003 );
4. [4] JW Mulholland, JC Shelton, XY Luo in "Journal of Fluids and Structures", Vol. 20, No. 1, pp. 129-140 (January 2005 );
5. [5] GE Engels, SLJ Blok, W. van Oeveren in "Biointerphases", Vol. 11, No. 3, 031004 (September 2016 );
6. [6] P. Borgdorff, RH van den Berg, MA Vis, GC van den Bos, GJ Tan Gelder in "The Journal of Thorac Cardiovascular Surgery", Vol. 118, No. 5, pp. 946-52 (1999 );
7. [7] AB Chandler in "Laboratory Investigation J Tech Methods Pathol." Vol. 7, No. 2, pp. 110-114 (1958 );
8. [8th] S. Braune, M. Grunze, A. Straub, F. Jung in "Biointerphases", 8:33, (December 2013 );
9. [9] S. Braune, A. Lendlein, F. Jung in "Hemocompatibility of Biomaterials for Clinical Applications", 1st edition, chapter: parts 1-3, pp. 51-76 (November 2017 );
10. [10] S. Sinn, T. Scheuermann, S. Deichelbohrer, G. Ziemer, HP Wendel in "Journal of Materials Science: Materials in Medicine", Vol. 22, No. 6, pp. 1521-1528 (June 2011 );
11. [11] WO 2005/033671 A1 ;
12. [12] JP 2007/089974 A ;
13. [13] US 4,627,419 A ;
14. [14] US 6,066,085 A ;
15. [15] US 2010/0204539 A1 ;
16. [16] US 6,579,223 B2 ; and
17. [17] M. Kilani et al. in “Sensors and Actuators” A 247 ( 2016 ) 440-447 .

The in-vitro investigation of the blood compatibility (BV) of cardiovascular implants and medical devices is an essential part of their development and optimization. Of particular interest are flow-dependent processes or flow-dependent parameters, such as the parameters hemolysis (dissolution of red blood cells or erythrocytes), activation of thrombocytes (platelet or platelet activation) or the degradation of von Willebrand factor (carrier protein of blood coagulation factor VIII), such as B. described in [1-3]. These processes are primarily determined by the geometric properties of a test object, in particular a cardiovascular implant, and the flow conditions in the blood vessel. In order to qualitatively and/or quantitatively record the effect of the test object on blood, shear rate-dependent influences should be suppressed as far as possible by the examination apparatus.

For very small test objects, e.g. cardiovascular implants such as stents (vascular supports), which have been implanted millions of times and are in constant development, there is interest in miniaturized test environments with small blood volumes. In small volumes, measurable markers, which are induced by flow-dependent processes in the blood, are present in a concentration that facilitates their determination. Known systems or pump mechanisms are only suitable to a limited extent for dynamic in-vitro examination of the interaction of blood with the test object, e.g. the stent, since they do not allow dynamic examination under controlled and physiological flow conditions and/or with sufficient reproducibility. The fluidic optimization of the geometry of a cardiovascular implant is therefore usually only carried out on the basis of numerical simulations. For example, in [4] and [5] the flow dynamics of two conventionally used test devices described in more detail below, the roller pump (squeeze pump) and the so-called "hemobile", are numerically examined for dynamic pressure changes.

Conventional devices for dynamic in-vitro testing of the blood compatibility or hemocompatibility of test objects, for example cardiovascular implants, are typically equipped with a tube in which the test object is firmly arranged and through which blood flows. Either the tube can be fixed stationary and a blood flow can be generated in the tube, or the blood flow can be generated by moving the tube.

In the technology described in [4], a conventional roller pump (squeeze pump) is used as the pump. The disadvantage is that rollers acting on a stationary tube induce shear forces, which damage the blood forced through the tube and thus falsify the test results on the blood compatibility of the test object.

The so-called "Chandler loop" described in [7, 10] is a plastic tube approximately half filled with blood, in which the test object is fixed and which is closed into a ring and set in rotation, whereby the test object is periodically drawn through the blood becomes. However, this technique has the disadvantage that the test object comes into contact with air with each revolution, in contrast to physiological conditions in the body. Contact with air can trigger reactions that distort the test result regarding blood damage caused by shear forces. In particular, due to the air space required in the system, foam formation and turbulence occur during rotation, which results in uncontrolled flow conditions and data that can only be reproduced to a limited extent and/or can only be generated with high scatter widths. In addition, the maximum achievable flow rate is severely limited by capillary forces, particularly with small test objects and/or with small tube diameters, which is why physiological flow rates cannot generally be applied for stents, for example. The capillary forces can cause the blood to move with the tube at too high a rotational speed, so that there is no longer any relative movement between the test object and the blood.

The "Hemobile" described in [1,5] is, like the "Chandler Loop", equipped with a closed hose, which, however, is set in rotation with a changing direction of rotation using a device described in [11]. With the "Hemobile" the tube is completely filled with blood, i.e. without any air space, and a ball valve is installed that prevents the blood from flowing back when the direction of rotation of the tube is reversed. The disadvantage is that the ball valve damages the blood flowing through it through shear forces and falsifies the test results for examining the interaction between blood and test object. Furthermore, as with the "Chandler Loop", the maximum achievable flow velocity is limited due to capillary forces with thin tubes.

[6] describes the creation of a flow due to hydrostatic forces or gravitational force by alternately raising and lowering the ends of a hose become. However, the flow speed generated by gravity is far too low to simulate physiological flows, given the usual height difference in the laboratory when raising and lowering the hose ends of, for example, 30 cm. Significantly larger height differences would require excessive blood volumes for examination.

In-vivo blood pumps are also known. These are divided into two classes for extracorporeal use in heart-lung machines, for example during heart surgery, and for implantation. In [12] the external pumping of blood using an impeller is described, which, however, generates shear forces that affect the blood. [13] describes extracorporeal pumping with a combination of gravity, which is created by rotating a fluid chamber partly filled with blood and partly with an inactive liquid, and the pumping of the inactive liquid. Implanted in-vivo blood pumps in [14-16] each generate lateral pressure on a membrane relative to the flow direction of the blood, which is done by means of a plate in [14] and fluid-driven in [15] and [16]. The in-vivo blood pumps described above are unsuitable for use as test devices for in-vitro examination of the interaction of blood with a test object.

The robustness and precision of conventional devices for in-vitro examination of interactions between blood and a test object are not sufficient to resolve subtle differences in the geometry or surface properties of test objects, in particular stents, under physiological flow conditions and, on this basis, to enable careful optimization of the To be able to carry out implant geometry. Furthermore - as summarized in [8,9] - there are no standards for in-vitro studies of the interaction of blood with test objects, which hinders the comparability of test results and the development of optimized blood-compatible products.

Not only cardiovascular implants, such as stents, but also medical devices, such as catheters, are of interest as test objects for in-vitro examinations.

From [17] a method for gently pumping liquids sensitive to shear stress is known, in which a piston is arranged in a liquid line and moved with an external magnetic field. The piston pushes the liquid in front of it in the liquid line.

The object of the invention is to provide an improved method and/or an improved device for in-vitro examination of the interaction of blood with a test object, with which disadvantages of conventional techniques are avoided. In particular, the change in blood components due to any shear forces in the device that could occur without the test object should be minimized. Alternatively or additionally, the task is to provide an easily implemented, parallelizable and/or cost-effective in-vitro examination of the interaction of blood with a test object, the use of which requires only small amounts of blood for the examination. Another task is to improve the in-vitro investigation of the interaction of blood with a test object so that precise measurement data with small spreads are obtained.

This object is achieved by a method and/or a device for in-vitro examination of the interaction of blood with a test object having the features of the independent claims. Advantageous embodiments and applications of the invention emerge from the dependent claims.

According to a first general aspect of the invention, the above object is achieved by a method for in-vitro examination of the interaction of blood with a test object. The method includes the step of providing the test object and the blood in a container device, wherein the test object is fixed in the container device and the blood is arranged as a blood column along a longitudinal direction (longitudinal extent) of the container device. Furthermore, the method includes generating a blood flow in the container device for a predetermined exposure time by means of a pressure generating device, wherein the blood flow passes the test object and/or passes through an opening in the test object. The method further includes examining the blood to determine at least one blood parameter that depends on an interaction of the blood with the test object.

The pressure generating device comprises at least one pressure generator with which an overpressure or a negative pressure can be generated in the container device relative to atmospheric pressure or relative to a further pressure generator connected to the container device. According to the invention, it is provided that a specifically adjustable, variable pressure is exerted on the blood column from the outside by the pressure generating device at at least one end of the container device, so that the blood flow is generated with an alternating flow direction along the longitudinal direction of the container device. The application of external pressure (excessive or negative pressure relative to atmospheric pressure) on the blood column means that the pressure applied by the pressure generating device acts on at least one end of the blood column in the container device.

According to a further general aspect, the above-mentioned task is solved by a device which is set up for in-vitro examination of the interaction of blood with a test object. The device comprises a container device which is designed to hold the test object to be examined in a fixed arrangement and a test volume of blood in the form of a blood column along a longitudinal direction of the container device. The device further comprises a pressure generating device which is designed to generate a blood flow in the container device, which flows past the test object and/or passes through an opening in the test object.

According to the invention, the container device has two ends (hereinafter also referred to as the first end or first ends and the second end or second ends), the pressure generating device being coupled in a pressure-tight manner to at least one of the ends. Furthermore, according to the invention, the pressure generating device is designed to exert an external, variable pressure on the blood column at at least one of the ends of the blood column, so that the blood flow can be generated with an alternating flow direction along the longitudinal direction of the container device.

With the pressure generation and application of pressure from the outside provided according to the invention, the pressure at at least one end of the blood column and with this the flow speed of the blood can advantageously be adjusted in a targeted manner. Deviating from conventional technology e.g. B. according to [6], in which the flow velocity of the blood is determined by the length of the blood column and can only be varied within a limited range, according to the invention a freely selectable external pressure is exerted, that is, there is an additional degree of freedom in setting the flow velocity of the blood Blood is available regardless of the length of the blood column and even with a small inner diameter of the container device. The flow velocity can be reproducibly adjusted over an extended range and with increased accuracy. The comparability of simultaneous or successive multiple measurements increases, and standards for characterizing blood compatibility can be defined, in particular based on the set flow speed and the inner diameter of the container device. Advantageously, the device according to the invention has a structure that can be implemented with inexpensive components.

Given the ever-increasing costs and case numbers of blood circulation diseases, blood compatibility tests will continue to be essential for the further development of cardiovascular implants and medical devices in the future. Based on the method according to the invention and the associated device for in-vitro examination of the interaction of blood with a test object, robust and easily parallelizable tests for their products can be provided, for example, for the approximately 100 stent-manufacturing companies worldwide. The advantage for users comes from the possibility of cost-effectively optimizing their products with regard to blood-compatible flow properties.

The container device comprises at least one object holding container, for example a plastic tube. The at least one object receptacle has an elongated shape with an opening at each end for exerting pressure and/or for pressure equalization. A blood column can be generated, for example, in a container device in the form of a substantially U-shaped tube. The ends of the blood column can be located in the two legs of the U-shaped tube that point upwards (opposite to the direction of gravity). If the ends of the at least one object-receiving container are suitably sealed, at least one object-receiving container with a straight shape or with a curved shape with ends pointing downwards (in the direction of gravity) can alternatively or additionally be used.

The container device or the object receiving container preferably comprises a non-stretchable material that is not deformed or is only deformed to a negligible extent by the external, variable pressure. Alternatively, the material can be described as not or only moderately elastic.

The blood can be provided, for example, from existing fresh blood and/or fresh blood taken separately from a test subject for other purposes or, optionally, from a blood bank. A small blood sample volume is used for the test, for example in the range of 0.5 ml to 10 ml. Taking the blood from the test subject is not part of the method according to the invention.

The container device advantageously has a length along the longitudinal extent which is at least long enough for the test object to be completely wetted by blood at any time during the blood flow with an alternating flow direction. The length of the container device is preferably between 20 cm and 100 cm. The inside diameter of the container device is preferably between 2 mm and 10 mm. The capacity of the container device is preferably between 0.5 ml and 10 ml.

The alternating external pressure generation at one or both ends of the blood column causes the blood inside the blood column to flow around and/or through the test object with low shear rates. The test object is advantageously wetted by blood at any time during the blood flow with an alternating flow direction. It is preferably provided that the test object is not brought into contact with air or a pressurized fluid during the period in which the blood flow is generated. By applying an external, variable pressure, physiological flow velocities, for example between 100 mm/s and 400 mm/s or beyond, but typically not greater than 2000 mm/s, can also be achieved. The pressure change at one or both ends of the object container or container device can be repeated at a fixed frequency, for example between 1 Hz and 2 Hz.

The process is preferably carried out at constant temperature. For example, the container device can be temperature-controlled in a thermal cabinet.

According to a preferred exemplary embodiment, the pressure is exerted alternately at both ends as excess pressure. By alternately applying excess pressure to both ends of the blood column, physiological flow velocities can advantageously be achieved in both flow directions relative to the test object. Furthermore, by alternately applying excess pressure at both ends, the period of the pressure change can advantageously be shortened compared to an excess pressure that can only be applied on one side. It is also advantageous that the alternating application of positive pressure avoids negative pressure, especially at the ends of the blood column, and thus minimizes outgassing and bubble formation.

Different types of pressure generators are advantageously available, which form the pressure generating device. Pressure generators include e.g. B. pressure fluid sources and/or pressure pistons. According to the invention, the pressure is exerted by the pressure generating device on at least one of the ends of the container device by applying a compressed gas, in particular air or a noble gas, and/or liquid fluid acting on the blood. In the exemplary embodiment with the alternating application of excess pressure at both ends of the blood column, two pressure pistons are provided. The pressure pistons and the blood column are through one Buffer separated, for example by the liquid or gaseous fluid that does not interact with the blood (inert fluid). The buffer is the above-mentioned compressed gas and/or the inert fluid whose density is lower than the density of blood. Due to the lower density, the inert fluid is located directly above the ends of the blood column. According to a further variant of the method and the associated device, a pressurized fluid source, such as. B. a compressed gas reservoir, with a valve control for generating an alternating overpressure can be provided at the two ends of the container device.

The container device may include a single object receiving container having a first and an opposite second end. Alternatively, the container device may comprise a plurality of object receiving containers, each having a first and an opposite second end. In a first exemplary embodiment with several object receptacles connected in parallel, the pressure generating device acts on all object receptacles together, so that the same pressure is exerted at the first or second ends of all object receptacles. In this case, the first ends and the second ends are each connected to one another, e.g. B. connected via a connecting line. In an alternative second exemplary embodiment with a plurality of object receptacles arranged next to one another, the pressure generating device acts on each object receptacle individually, so that individual pressure is exerted on each object receptacle. The length, diameter and volume of each object holding container can correspond to the above-mentioned sizes of the container device.

The pressure generator of the pressure generating device in the form of the pressure piston can comprise at least one piston syringe. In the case of object receptacles connected in parallel, the piston syringe according to a first exemplary embodiment acts simultaneously on the first ends of all object receptacles. In the exemplary embodiment with the alternating application of excess pressure at both ends of the container device, a first piston syringe and a second piston syringe are provided, each of which acts accordingly on the first and second ends of the container device. According to a further exemplary embodiment, the pressure generating device can comprise a plurality of first and/or second piston syringes, each of which acts individually on the first and/or the second ends of the object receiving containers.

The test object can be, for example, a cardiovascular implant or a medical device. The implant can in particular be a stent. The medical product can in particular be a catheter.

The exposure time (duration of generation of a blood flow) is advantageously at least one hour and/or at most six hours. The exposure time is particularly preferably chosen in the range of two to three hours. An exposure time of at least one hour eliminates short-term effects, in particular a reduced function of platelets, which are caused by the transfer of the blood into the container device - in particular a container device without a test object. Such short-term effects would distort the test results with regard to the interaction of blood with the test object. Furthermore, the exposure time should not exceed six hours, as after this period at the latest, changes in blood outside of a living body usually occur and this results in a falsification of the test results. The maximum exposure time of the device according to the invention exceeds the maximum exposure time of four hours of conventional devices, e.g. the "Chandler loop" or the roller pump (squeeze pump), whose air contact and / or mechanical action due to lateral pressure on the tube damages the blood components at an early stage.

The at least one blood parameter, which is preferably recorded after the exposure time, is preferably at least one of the parameters hemolysis, degradation of von Willebrand factor and activation of platelets. Alternatively or additionally, a blood count (hemogram), for example a small or large one, can be created. The blood count may contain a number of leukocytes, erythrocytes, hemoglobin, platelets and non-specific inflammatory markers. Alternatively, the proportion of hematocrit and/or the mean platelet volume can be determined.

According to a further variant of the invention, the method can include the step of examining the test object to determine a coverage density of the test object with blood components after the exposure time. The coverage density with blood components can e.g. B. are formed by an attachment of platelets and/or proteins. After completion of the procedure, the test object can be examined outside the container device using staining methods and/or spectroscopy. Alternatively or additionally, the test object and/or the blood can be examined in situ during the exposure time using a camera and/or a microscope and/or using immunobiochemical methods.

According to a further preferred exemplary embodiment, the container device, in particular the at least one object receiving container, has an internal cross-sectional dimension transverse to the longitudinal extent, which is adapted to an external cross-sectional dimension of the test object. A cardiovascular implant, such as B. a stent, can be introduced into the object receptacle in a folded state and unfolded therein with a balloon catheter, so that the implant is fixed to the walls of the object receptacle. Alternatively or additionally, the test object can be placed in the container device or the at least one object receiving container by means of a holding device, such as. B. a needle inserted from the outside through the wall of the object container. The container device can be set up to hold the test object in the form of a cardiovascular implant or a medical product.

The container device or the at least one object receiving container preferably comprises a straight or U-shaped hose or a straight or U-shaped tube, in particular made of a plastic. The cross section of the at least one object holding container can be round, oval or polygonal. The U-shape of the at least one object receptacle with upwardly directed legs of the U can ensure that the blood column is held by gravity within the container device or the object receptacle. The material of the at least one object receptacle is preferably blood-compatible. Alternatively or additionally, the at least one object receptacle can be coated with a blood-compatible material. Blood-compatible material is characterized by the fact that blood remains unchanged or is changed negligibly little when it comes into contact with the material, and it includes e.g. B. a plastic, such as polyvinyl chloride (PVC) or silicone, and / or a surface coating made of heparin, polyethylene glycol (PEG) or zwitterionic molecules (e.g. sulfobetaines).

The at least one object receiving container preferably comprises a moderately elastic or an inelastic material. This material is preferably chosen so that propulsion of the blood column is not influenced or only slightly influenced, for example by less than 10%, by an expansion of the object receptacle wall. The inelastic or moderately elastic material of the at least one object receiving container can also be flexible, so that bending is possible for introducing the test object and for connecting at least one end to a pressure generating device.

Figur 1

eine schematische Teilseitenansicht eines Ausführungsbeispiels einer Vorrichtung zur invitro Untersuchung der Wechselwirkung von Blut mit einem Testobjekt;

Figur 2

eine perspektivische Teilansicht eines alternativen Ausführungsbeispiels der erfindungsgemäßen Vorrichtung mit mehreren Objektaufnahmebehältern;

Figur 3

eine perspektivische Teilansicht eines weiteren alternativen Ausführungsbeispiels der erfindungsgemäßen Vorrichtung mit mehreren Objektaufnahmebehältern;

Figur 4

eine schematische Ansicht eines Objektaufnahmebehälters mit einem Testobjekt in einer ersten Betriebsphase;

Figur 5

eine schematische Ansicht des Objektaufnahmebehälters mit einem Testobjekt gemäß Figur 4 in einer zweiten Betriebsphase; und

Figur 6

eine schematische Darstellung einer Druckerzeugungseinrichtung.

Further details and advantages of the invention are described below with reference to the accompanying drawings. Show it:

Figure 1

a schematic partial side view of an exemplary embodiment of a device for in vitro examination of the interaction of blood with a test object;

Figure 2

a partial perspective view of an alternative exemplary embodiment of the device according to the invention with several object holding containers;

Figure 3

a partial perspective view of a further alternative exemplary embodiment of the device according to the invention with several object holding containers;

Figure 4

a schematic view of an object receptacle with a test object in a first operating phase;

Figure 5

a schematic view of the object receptacle with a test object according to Figure 4 in a second operational phase; and

Figure 6

a schematic representation of a pressure generating device.

The Figures 1 to 3 show schematic partial views of a device 10 according to the invention for in-vitro examination of the interaction of blood with a test object. Other components of the device 10, such as. B. a control device, in particular for controlling the pressure generator of the pressure generating device, or details of examination devices 50, 51 are not illustrated.

The device 10 comprises a container device 12 and a pressure generating device 40 with at least one pressure generator (e.g. the illustrated piston syringe) at least at a first end 16 of the container device 12. In the exemplary embodiment according to Figure 1 the container device 12 includes a single object receiving container 14 in which a blood column 20 is located. The object receptacle 14 is preferably bent in a U-shape with the first and second ends 16 and 17 pointing upwards. The blood column 20 is held in the center of the object receptacle 14 by gravity. The object receiving container 14 preferably comprises a blood-compatible and moderately elastic material. For example, the object receptacle made of polyvinyl chloride (PVC) or silicone. The pressure generating device 40, which is connected in a pressure-tight manner to the first end 16 of the object holding container 14, is designed to generate excess pressure. A further pressure generator of the pressure generating device (in Figure 1 not shown), which is designed to generate an overpressure at the second end 17.

According to an exemplary embodiment of the method for in-vitro examination of the interaction of blood with a test object, a test object 30, for example a cardiovascular implant (see Figures 4 and 5 ), introduced into an object receiving container 14 of a container device 12 and fixed. For example, after insertion into the object receptacle 14, a stent is unfolded by a balloon catheter and pressed against the walls of the object receptacle 14. According to a further exemplary embodiment, the introduced test object 30 is fixed in the object receiving container 14 by a holding needle. With a substantially U-shaped tube, the cardiovascular implant is preferably fixed centrally between the first and second ends of the object receptacle 14.

The object receiving container 14 or the container device 12 is filled with blood, so that a column of blood 20 can flow completely through and/or around the test object 30. According to the invention, the test object 30 has no contact with the ambient air, with a compressed gas 46 or with an inert fluid otherwise used as a buffer for the pressure generating device during the exposure time.

An overpressure (arrow 42 in.) is created at the first end 16 of the object holding container 14 at a first point in time Figure 4 ) generated by the pressure generating device 40. The excess pressure causes the blood column 20 to be driven from the first end 16 to the second end 17 of the object receptacle 14. At a second time, a second pressure generator of the pressure generating device at the second end 17 exerts an overpressure 42 on the blood column 20, so that the blood column 20 is driven (back) from the second end 17 to the first end 16 (arrow 42 in Figure 5). The alternating application of overpressure to the first end 16 and the second end 17 of the object receptacle 14 is repeated periodically at a fixed frequency, for example between 1 Hz and 2 Hz. The object receiving container 14, for example made of PVC, is filled with so much blood that the blood column 20 created in the tube corresponds to a multiple of the length of the test object 30. The test object 30 is completely wetted with blood at any time during a flow period.

When the pressure generating device 40 is in operation, there is no mechanical deformation of components of the device that are in contact with blood, as a result of which the blood located in the object receiving container 14, for example a hose, is gently pushed over the fixed test object 30 without any shear rates. The flow speed of the blood can be determined by the level of the pressure difference, in particular the excess pressure applied alternately at the first end 16 and at the second end 17 of the object receptacle 14 (arrow 42 in Figures 4 and 5 ), be regulated on both sides.

The cycle or periodic pressure generation is repeated until the desired exposure time of at least one hour and at most six hours, for example between two and three hours, is reached. Relevant blood parameters such as hemolysis, the degradation of von Willebrand factor and/or the activation of platelets are then determined using an examination device 50. The schematically shown examination device 50 can be used for various examination methods or assays, such as. B. immunobiochemical assays, spectroscopic analyzes and / or microscopic analyzes can be designed.

In a further advantageous embodiment, the coverage density of the test object 30 with blood components is determined using staining techniques and/or spectroscopic methods. Alternatively or additionally, in a further advantageous embodiment, blood components can be deposited on the test object 30 during the exposure by means of a further examination device 51 (see Figure 1 ), for example a camera and/or a spectroscope.

Figure 2 shows a further exemplary embodiment of a device 10 for in-vitro examination of the interaction of blood with a test object, in which the container device 12 comprises a plurality of object receptacles 14 connected in parallel. According to the in Figure 2 In the illustrated embodiment, the device comprises a pressure generating device 40 with a first pressure generator which acts in parallel on the first ends 16 of the plurality of object receiving containers 14. Alternatively or additionally, a second pressure generator (not shown) of the pressure generating device can act in the same way on the second ends 17 of the object receiving containers.

Figure 3 shows a further exemplary embodiment of a device 10 for in-vitro examination of the interaction of blood with the test object, in which the container device 12 comprises a plurality of object receiving containers 14 arranged next to one another and each object receiving container 14 is connected at its first end 16 to an individually controllable pressure generator of the pressure generating device 40 . Alternatively or additionally, further pressure generators (not shown) of the pressure generating device can be connected to the second ends 17 of the object receiving containers 14.

The exemplary embodiments of the Figures 2 and 3 can be combined, for example, by connecting the first ends 16 with a single pressure generator (e.g. pressure piston) acting on all object receptacles 14 and each of the second ends 17 with a single pressure generator (e.g. pressure piston) acting on the associated object receptacle 14 ) connected is.

Figure 4 and Figure 5 show schematically an object holding container 14 of a container device 12 with a test object 30. The object holding container 14 extends in practice, as in the Figures 1 to 3 shown, preferably U-shaped with upwardly directed ends 16, 17. In practice, however, the illustrated straight shape of the object receiving container 14 can also be provided. The object receptacle 14 has a length 18 of z. B. 70 cm and an inner diameter 19 of z. B. 3 mm. The object holding container 14 is in the Figures 4 and 5 filled with a blood column 20 and a test object 30 is fixed essentially centrally in the object receiving container 14. According to this exemplary embodiment, there is a compressed gas 46 at the first and second ends 16 and 17 of the object receiving container 14. Alternatively or additionally, the blood column 20 can be buffered by another inert fluid 46 with respect to a pressure generator of a pressure generating device 40.

In Figure 4 At the beginning of a first operating state of the device 10, the blood column 20 is located close to the first end 16 of the object receiving container 14. A pressure generator (not shown) of the pressure generating device at the first end 16 generates an overpressure in the compressed gas 46 (see arrow 42) around the blood column 20 to drive to the second end 17. At the end of the first operating state there is the blood column 20, as in Figure 5 shown, near the second end 17 of the object receptacle 14. In a second operating state of the device 10, a second pressure generator (not shown) of the pressure generating device at the second end 17 now generates an overpressure in the compressed gas 46 (see arrow 42), which brings the blood column 20 to the first drifts back at the end of 16. As soon as the blood column 20 is again close to the first end 16, as in Figure 4 shown, the device 10 is switched back to the first operating state.

Figure 6 shows an exemplary embodiment of a pressure generating device 40, which is arranged at a first end 16 of an object holding container 14. The pressure generating device 40 comprises a pressure piston 44, which generates an excess pressure (see arrow 42) on a compressed gas 46 or other inert fluid located in a closed cavity. Alternatively or additionally, a further, preferably identical, pressure generating device can be arranged at a second end 17 of the object receiving container 14.

The pump mechanism according to the invention for dynamic tests for the interaction of blood with the test object 30 advantageously produces a low-shear rate propulsion of the blood over the test object 30. Furthermore, the method according to the invention advantageously enables examinations with very small sample volumes, for example small amounts of blood between 0.5 ml and 10 ml small diameters of the test object 30. The influence of the geometry of the test object 30, for example an implant, on flow-dependent blood reactions can be mapped precisely and robustly.

At the same time, physiological flow profiles (i.e. high flow rates and/or pulsed flow) can be applied in a controlled manner, which forms the basis for the collection of robust, reproducible and therefore usable data. In addition, the mechanism is simple and inexpensive, can be easily parallelized, and can be flexibly adapted to the respective sample geometry, for example the geometry of the test object 30. This means it differs significantly from the state of the art that has been used for decades with the restrictions described above, e.g. the “Chandler loop”, the roller pump (squeeze pump) and/or the “Hemobile”.

Although the invention has been described with reference to exemplary embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents substituted. Furthermore, many modifications can be made to adapt a particular container device or a particular pressure generating device to the teachings of the invention. Accordingly, the invention is not limited to the disclosed embodiments, but includes all embodiments that fall within the scope of the appended claims.

The features of the invention disclosed in the above description, the drawings and the claims may be important both individually and in combination or subcombination for the realization of the invention in its various embodiments.

Claims (15)

1. Method for in vitro investigating the interaction of blood with a test object (30), comprising the steps of

   - providing the test object (30) and the blood in a container device (12), wherein the test object (30) is fixed in the container device (12) and the blood is arranged as a blood column (20) along a longitudinal direction (18) of the container device (12),

   - generating a blood flow in the container device (12) for a predetermined exposure time by means of a pressure generating device (40), the blood flow passing the test object (30) and/or passing through an opening of the test object (30), and

   - investigating the blood to determine at least one blood parameter which is dependent on an interaction of the blood with the test object (30), wherein

   - an external, variable pressure is exerted on the blood column (20) with the pressure generating device (40) at at least one end (16, 17) of the container device (12), so that the blood flow is generated with an alternating flow direction along the longitudinal direction (18) of the container device (12), characterized in that

   - the pressure is exerted by the pressure generating device (40) at at least one of the ends (16, 17) of the container device (12) by an application of a pressurized gas (46) acting directly on the blood and not interacting with the blood and/or a liquid fluid (46) acting directly on the blood and not interacting with the blood, the density of which is lower than the density of the blood.
2. Method according to claim 1, wherein the pressure is alternately applied to both ends (16, 17) of the container device (12) as an overpressure.
3. Method according to one of the preceding claims, in which

   - the pressure is exerted by the pressure generating device (40) by the application of the pressurized gas (46) acting on the blood, the pressurized gas (46) comprising air or a noble gas.
4. Method according to one of the preceding claims, in which the container device (12) comprises

   - a single object receiving container (14), or

   - multiple object receiving containers (14), wherein the pressure generating device (40) acts on all object receiving containers (14) together or on each object receiving container (14) individually.
5. Method according to one of the preceding claims, comprising at least one of the features

   - the test object (30) is a cardiovascular implant or a medical device, and

   - the exposure time is at least one hour and/or at most six hours.
6. Method according to one of the preceding claims, wherein

   - the at least one blood parameter is at least one of hemolysis, degradation of von Willebrand factor and activation state of thrombocytes.
7. Method according to one of the preceding claims, comprising the step of

   - Investigating the test object (30) to determine an occupancy density with blood components, in particular an accumulation of thrombocytes and/or proteins.
8. Device (10) being adapted for in vitro investigating of the interaction of blood with a test object (30), comprising

   - a container device (12) configured to receive the test object (30) to be examined in a fixed arrangement and a test volume of blood in the form of a blood column (20) along a longitudinal direction (18) of a container device (12), and

   - a pressure generating device (40) configured to generate in the container device (12) a flow of blood passing the test object (30) and/or passing through an opening of the test object (30), wherein

   - the container device (12) has two ends (16, 17), wherein the pressure generating device (40) is coupled to at least one of the ends (16, 17), and

   - the pressure generating device (40) is configured to exert an external, variable pressure on the blood column (20) at at least one of the ends of the container device (12), such that the blood flow can be generated with an alternating flow direction along the longitudinal direction (18) of the container device (12)
   characterized in that

   - the pressure generating device (40) is configured to exert the pressure at at least one of the ends (16, 17) of the container device (12) by an application of a pressurized gas (46) acting directly on the blood and/or a liquid fluid (46) acting directly on the blood and not interacting with the blood, the density of which fluid is lower than the density of the blood.
9. Device (10) according to claim 8, in which

   - the pressure generating device (40) is configured to exert the pressure as an overpressure alternately at both ends (16, 17) of the container device (12).
10. Device (10) according to claim 8 or 9, in which

    - the pressure generating device (40) is configured to exert the pressure by the application of the pressurized gas (46) acting on the blood, the pressurized gas (46) comprising air or a noble gas.
11. Device (10) according to one of claims 8 to 10, in which

    - the container device (12) has a length in the longitudinal extension (18) at least long enough to wet the test object (30) with blood at any time of the blood flow with alternating flow direction.
12. Device (10) according to one of claims 8 to 11, wherein the container device (12) comprises

    - a single object receiving container (14), or

    - a plurality of object receiving containers (14), wherein the pressure generating device (40) acts on all object receiving containers (14) together or on each object receiving container (14) individually.
13. Device (10) according to one of claims 8 to 12, wherein

    - the container device (12) or the at least one object receiving container (14) has, transversely to the longitudinal extension (18), an internal cross-sectional dimension (19) adapted to an external cross-sectional dimension of the test object (30).
14. Device (10) according to one of claims 8 to 13, wherein

    - the test object (30) is fixed in the container device (12) or the at least one object receiving container (14) by means of a retaining device.
15. Device (10) according to one of claims 8 to 14, having at least one of the features

    - the container device (12) is adapted to receive the test object (30) in the form of a cardiovascular implant or a medical device, and

    - the container device (12) or the at least one object receiving container (14) comprises a straight or U-shaped bent hose or a straight or U-shaped bent tube, in particular made of a plastic.

Images